Soil bacteria of the genus Rhizobium, a member of the family Rhizobiaceae, are capable of infecting plants and inducing a highly differentiated structure, the root nodule, within which atmospheric nitrogen is reduced to ammonia by the bacteria. The host plant is most often of the family Leguminosa. Previously, Rhizobium species were informally classified in two groups, either as "fast-growing" or "slow-growing" to reflect the relative growth rates in culture. The group of "slow-growing" rhizobia has recently been reclassified as a new genus, Bradyrhizobium (Jordan, D. C. (1982) International Journal of Systematic Bacteriology 32:136). The fast-growing rhizobia include Rhizobium trifolii, R. melliloti, R. leguminosarum and R. phaseolus. These strains generally display a narrow host range. Fast-growing R. japonicum which nodulate Glycine max cv. Peking and fast-growing members of the cowpea Rhizobium display broader host range. The slow-growing rhizobia, a distinct genus now called Bradyrhizobium, include the commercially important soybean nodulating strains Bradyrhizobium japonicum (i.e.; USDA 110 and USDA 123), the symbiotically promiscuous rhizobia of the "cowpea group", and Bradyrhizobium sp. (Parasponia) (formerly Parasponia Rhizobium) which nodulates the non-legume Parasponia, as well as a number of tropical legumes including cowpea and siratro.
Nodulation and development of effective symbiosis is a complex process requiring both bacterial and plant genes. Several recent reviews of the genetics of the Rhizobium-legume interaction are found in Broughton, W. J., ed. (1982) Nitrogen Fixation, Volumes 2 and 3 (Clarendon Press, Oxford); Puhler, A. ed. (1983) Molecular Genetics of the Bacteria-Plant Interaction (Springer-Verlag, Berlin); Szalay, A. A. and Leglocki, R. P., eds. (1985) Advances in Molecular Genetics of the Bacteria-Plant Interaction (Cornel 1 University Publishers, Ithaca, N.Y.); Long, S. R. (1984) in Plant Microbe Interactions Volume 1, Kosuge, T. and Nester, E. W. eds. (McMillan, N.Y.) pp. 265-306; and Verma, D. P. S. and Long, S. R. (1983) International Review of Cytology (Suppl. 14), Jeon, K. W. (ed.), Academic Press, p. 211-245.
In the fast-growing species, the genes required for nodulation and nitrogen fixation are located on large Sym (symbiotic) plasmids. Although the process of recognition, infection and nodule development is complex, it appears that at least for the fast-growing rhizobia relatively few bacterial genes are directly involved and these are closely linked on the Sym plasmid. For example, a 14 kb fragment of the Rhizobium trifolii Sym plasmid is sufficient to confer clover-specific nodulation upon a Rhizobium strain cured of its Sym plasmid, as well as on an Agrobacterium strain which does not normally nodulate plants (Schofield et al., (1984) Plant Mol. Biol. 3:3-11). Nodulation and nitrogenase genes are localized on symbiotic plasmids in R. leguminosarum (Downie et al. (1983) Mol. Gen. Genet. 190:359-365) and in R. meliloti (Kondorosi et al. (1984) Mol. Gen. Genet. 193:445-452). In contrast, no Sym plasmids have been associated with the slow-growing rhizobia, B. japonicum or Bradyrhizobium sp. (Parasponia). The nitrogenase and nodulation genes of these organisms are believed to be encoded on the chromosome.
Fine structure genetic mapping has been used to locate individual nodulation genes in fast-growing rhizobia. Transposon mutagenesis, most often using the transposon Tn5, has identified about 10 nodulation genes associated with non-nodulation, delayed nodulation and altered host range phenotypes (Djordjevic et al. (1985) Mol. Gen. Genet. 200:263-271; Downie et al. (1985) Mol. Gen. Genet. 198:255-262; Kondorosi et al., 1984, and Innes et al. (1985) Mol. Gen. Genet. 201:426-432). Common nodulation genes designated nodABC and D, which are functionally and structurally conserved among the fast-growing rhizobia, have been identified by hybridization studies and cross-species complementation experiments (Banjalvi et al. (1981) Mol. Gen. Genet. 184:318-325; Djordjevic et al. (1985) Plant Mol. Biol. 4:147-160; Kondorosi et al. (1984) Mol. Gen. Genet. 193:445-452; and Fisher et al. (1985) Applied Environ. Microbiol. 49:1432-1435). Both Bradyrhizobium sp. (Parasponia) (Marvel et al. (1985) Proc. Natl. Acad. Sci. USA 82:5841-5845) and B. japonicum (Russel et al. (1985) J. Bacteriol. 164:1301-1308) contain nodulation genes which can functionally complement mutations in fast-growing rhizobia and which show strong structural homology to nodulation gene regions of R. meliloti and R. leguminosarum. In addition to the common nod genes, adjacent regions (IV and V) which are involved in host specificity of nodulation (nodG and nodH) have been identified in R. meliloti (Kondorosi et al. 1984) and R. trifolii (Djordjevic et al. 1985; Rolfe et al. (1985) Nitrogen Fixation Research Progress, Evans et al. (eds.), Martinus Nijhoff, pp. 79-85.
DNA sequencing of the common nod gene region has revealed similarities in organization of these nod genes in fast growing rhizobia, as shown in FIG. 1. The open reading frames of nodA, B and C are grouped sequentially and believed to be coordinately transcribed as a single transcriptional unit. The nodD open reading frame reads divergently from the nodABC operon (Egelhoff et al. (1985) DNA 4:241-248; Jacobs et al. (1985) J. Bacteriol. 162:469-476; Rossen et al. (1984) Nucleic Acids Res. 12:9497-9508; and Torok et al. (1984) Nucl. Acids Res. 12:9509-9524; Schofield (1985) Nitrogen Fixation Research Progress, Evans et al. (eds.) Martinus Nijhoff, p. 125; and Rolfe et al., ibid., 1985). The DNA sequence between nodA and nodD is presumed to contain divergent promoters for each of these regions.
Sym plasmid encoded nodulation genes of Rhizobium strains and their analogues in Bradyrhizobium strains affect the early stages of nodule formation including host-bacterium recognition, infection and nodule development. Among strains that infect a particular host, there is some variation in the rates of initiation of nodulation which is reflected ultimately in differences in competitiveness between strains for nodule occupancy on the host. Strains which initiate infection and nodules earlier will occupy a greater portion of the nodules on a given plant. The timing of the initiation of nodulation by a strain appears to be genetically determined. Improving the competitiveness of a specific Rhizobium is an important part of the development of improved inocula for legumes. A more effective Rhizobium strain, which would likely constitute an improved inoculum, must be able to out-compete the indigenous rhizobia population for nodule occupancy in order for their improved qualities to impact on the inoculated legume.
The precise biochemical role of the nod genes and their products in nodule development is unknown. Attempts to isolate nod gene mRNA and protein products from free-living Rhizobium have been unsuccessful (Kondorosi et al. 1984). Protein products of several nod genes have, however, been obtained by fusion of nod genes to strong E. coli promoters (Schmidt et al. (1984) EMBO J. 3:1705-1711; and John, M. et al. (1985) EMBO J. 4:2425-2430) or in an E. coli in vitro transcription/translation system (Downie et. al. (1985) Mol. Gen. Genet. 198:255-262).
The establishment of nitrogen-fixing nodules is a multistage process involving coordinated morphological changes in both bacterium and plant, so it is expected that the rhizobial nodulation genes are under precise regulatory control. It has been suggested that an exchange of signals between plant and bacterium is requisite for mutual recognition and coordination of the steps of infection and nodulation development (Nutman, P. S. (1965) in Ecology of Soil Borne Pathogens, eds. F. K. Baker and W. C. Snyder, University of California Press, Berkeley, pp. 231-247; Bauer, W. D. (1981) Ann. Rev. Plant Phys. 22:407-449; and Schmidt, E. E. (1979) Ann. Rev. Microbiol. 33:355-376). For example, legume exudates have been linked to control of nodulation. Exudates have been reported to both stimulate (Thornton (1929) Proc. Royal Soc. B 164:481; Valera and Alexander (1965) J. Bacteriol. 89:113-139; Peters and Alexander (1966) Soil Science 102:380-387) or inhibit (Turner (1955) Annals Botany 19:149-160; and Nutman (1953) Annals Botany 17:95-126) nodulation by rhizobia.
Recently, Baghwat and Thomas (1982) Applied Environ. Microbiol. 43:800-805 described a stimulatory factor from legume exudates that was thermostable, was high molecular weight (about 2.times.10.sup.5) and was composed of protein and neutral hexoses. This factor was associated with elimination of nodulation delay in a certain cowpea Rhizobium strain. Halverson and Stacey (1984) Plant Physiol. 74:84-89; and (1985) Plant Physiol. 77:621-624 reported an exudate factor having a similar affect on nodulation initiation in B. japonicum USDA 110 mutants. In contrast to Baghwat and Thomas (1982), this stimulator of nodulation is described as a heat and trypsin sensitive protein; a galactose, specific lectin. Diverse chemicals have been identified as stimulators or inhibitors of nodulation. Reported stimulators of nodulation include inositol, indole, 2-phenol-n-butyric acid, D-leucine, barbituric acid, pyridine-3-sulfonate and quercetin (Molina and Alexander (1967) Can. J. Microbiol. 13:819-827; and Weir (1960) Phyton 15:109-118).
The chemical factors in legume exudates that are responsible for stimulation of nod gene expression have been identified. U.S. Patent Application Serial No. 844,870 filed Mar. 27, 1986, identified a structural related class of molecules, certain substituted flavones and flavanones as nod gene inducing factors. Individual purified molecules, either isolated from clover exudates or available from commercial sources, were found to induce expression of certain nod genes.